A major aspect of the pharmacology of drugs is the modification of their action and duration of action via biotransformation and excretion. Sulfation is one of the major pathways for the biotransformation and excretion of drugs as well as endogenous compounds such as steroids and catecholamines. The long-term objective of this research is to understand the role of sulfation in the metabolism of drugs and endogenous substrates in humans. As observed for other drug metabolizing enzymes such as the glutathione S-transferases and UDP-glucuronyltransferases, sulfotransferases, the enzymes catalyzing the transfer of the sulfate moiety to an acceptor compound, represent a family of distinct isoenzymes. However, in humans the number, characteristics and relationships of these enzymes are not well known. The research proposed in this application is directed toward investigating the protein chemistry and molecular biology of human phenol sulfotransferases. Human blood platelets, provide an accessible source of two forms of phenol sulfotransferase (PST) activity, the phenol (P) and catecholamine (M) PST activities. Investigation of the platelet PSTs may provide valuable insights into the role of sulfation in less accessible human tissues. Platelet PST activity may also serve as an indicator for the whole body metabolism of a given drug by sulfation. The goals of the proposed research are designed to investigate the biochemistry of the PSTs at the levels of their enzymic activity, protein chemistry, and molecular biology. First, the P- and M-PST activities in platelets will be purified and characterized. Purification techniques will include ChromatofocusingR, affinity chromatography, and specific antibody recognition. The P and M PST activities in platelets are believed to be catalyzed by different enzymes and are also very similar kinetically to PST activities in other tissues and organs indicating that these two enzymes are widely distributed in the body. Second, specific antibodies will be raised in rabbits against the purified PSTs. These antibodies will be used to identify and immunoquantitate the presence of PST proteins. Third, cDNAs encoding the two enzymes will be cloned. Antibodies raised against the two purified PSTs will be used to isolate the respective cDNAs from lambda gtll cDNA libraries. Since platelets are enucleated, lambda gtll libraries generated from human brain and liver will be used. cDNAs obtained from these libraries will be characterized so that they may serve as probes to study the regulation, heterogeneity, and structure of human PSTs.